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. Author manuscript; available in PMC: 2020 Apr 8.
Published in final edited form as: Dev Cell. 2019 Feb 28;49(1):130–144.e6. doi: 10.1016/j.devcel.2019.01.027

Figure 4. Phosphorylation of Stx17 by TBK1 is required for formation of ATG13 and FIP200 puncta.

Figure 4.

(A,B) HC analysis of effect of complementation of Stx17KO cells with FLAG-Stx17WT, FLAG-Stx17S202A and FLAG-Stx17S202D on formation of ATG13 dots in response to autophagy induction by incubation with EBSS for 1h. White masks, computer-identified FLAG positive cells (primary objects); red masks, computer-identified ATG13 dots in FLAG transfected cells. HeLa WT (left) were left un-transfected for control. (Scale bar 10 μm). **, p < 0.01, (n=3) ANOVA. (C,D) HC analysis to analyze the effect of complementation of Stx17KO with FLAG-Stx17 WT, FLAG-Stx17S202A and FLAG-Stx17S202D on formation of FIP200 dots in response to autophagy induction by incubation with EBSS for 1h. White masks, computer-identified FLAG positive cells (primary objects); red masks, computer-identified FIP200 dots in FLAG transfected cells. Scale bar 10 μm. **, p < 0.01, (n=3) ANOVA. (E,F). HC analysis of effect of cross-complementation of TBK1KO cells with FLAG-Stx17WT, FLAG-Stx17S202A and FLAG-Stx17S202D on formation of FIP200 dots in response to autophagy induction by incubation with EBSS for 1h. White masks, computer-identified FLAG positive cells (primary objects); red masks, computer-identified ATG13 dots in FLAG transfected cells.White masks, computer-identified FLAG positive cells (primary objects); red masks, computer-identified ATG13 dots in FLAG transfected cells. Scale bar 10 μm. **, p < 0.01, (n=3) ANOVA. (G,H) HC analysis of effect of cross-complementation of TBK1KO cells with FLAG-Stx17WT, FLAG-Stx17S202A and FLAG-Stx17S202D on formation of FIP200 dots in response to autophagy induction by incubation with EBSS for 1h. White masks, computer-identified FLAG positive cells (primary objects); red masks, computer-identified ATG13 dots in FLAG transfected cells. Scale bar 10 μm. **, p < 0.01, (n=3) ANOVA. (I) A model depicting the effect of Stx17 on formation of mammalian pre-autophagosomal structures and omegasomes. In HC experiments (A-H), Images are details from a large database of machine-collected and computer-processed images; data are from 3–5 independent experiments (>500 primary object examined per well; minimum number of wells, 12). See also Figure S4.